CN109790532A - Anti-lag-3 antibody - Google Patents

Anti-lag-3 antibody Download PDF

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Publication number
CN109790532A
CN109790532A CN201780050285.1A CN201780050285A CN109790532A CN 109790532 A CN109790532 A CN 109790532A CN 201780050285 A CN201780050285 A CN 201780050285A CN 109790532 A CN109790532 A CN 109790532A
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antibody
chain
amino acid
sequence number
acid sequence
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CN109790532B (en
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今内觉
大桥和彦
村田史郎
冈川朋弘
西森朝美
前川直也
铃木定彦
中岛千绘
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Hokkaido University NUC
Fuso Pharmaceutical Industries Ltd
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Hokkaido University NUC
Fuso Pharmaceutical Industries Ltd
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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Abstract

The present invention provides a kind of anti-lag-3 antibody that the animal for other than rat can also apply repeatedly.The present invention provides following anti-lag-3 antibody, it includes (a) L chains and (b) H chain, the L chain is with the CDR1 comprising the amino acid sequence with QSLLDSDGNTY (sequence number 16), the L chain variable region of the CDR3 of the CDR2 of amino acid sequence with SVS and the amino acid sequence with MQATHVPFT (sequence number 17), with the L chain constant region of the animal's antibody other than rat, the H chain is with the CDR1 comprising the amino acid sequence with GFDFDTYP (sequence number 18), the H chain variable region of the CDR3 of the CDR2 of amino acid sequence with ITIKTHNYAT (sequence number 19) and the amino acid sequence with NREDFDY (sequence number 20) and the H chain of the animal's antibody other than rat Constant region.It includes medical composition of the anti-lag-3 antibody as effective component that the present invention, which provides,.The present invention also provides the methods for manufacturing the anti-lag-3 antibody.

Description

Anti-lag-3 antibody
Technical field
The present invention relates to a kind of anti-lag-3 antibody, more specifically, are related to having comprising the anti-ox LAG-3 antibody of rat Complementary strand determines the anti-lag-3 antibody of the constant region of the variable region of area (CDR) and the antibody of the animal other than rat.
Background technique
Immunosupress receptor lymphocyte activationgene 3 (LAG-3) is accredited as nearly fate of CD4 (non-patent literature 1:Triebel F, Jitsukawa S, Baixeras E, Roman-Roman S, Genevee C, Viegas- Pequignot E, Hercend T.J.Exp.Med., 171 (5): 1393-1405；May 1,1990.), it has shown that in recent years Immunosupress (non-patent literature 2:Blackburn SD, Shin H, HainingWN, Zou in participation chronic infection disease, tumour T, Workman CJ, Polley A, Betts MR, Freeman GJ, Vignali DA, Wherry EJ.Nat.Immunol., 10 (1): 29-37；Nov.30,2008., non-patent literature 3:Woo S-R, Turnis ME, GoldbergM V., Bankoti J, Selby M, Nirschl CJ, Bettini ML, Gravano DM, Vogel P, Liu CL, Tangsombatvisit S, Grosso JF, Nettog, Smeltzer MP, Chaux A, Utz PJ, Workman CJ, Pardoll DM, Korman AJ, Drake CG, Vignali DAA.Cancer Res., 72 (4): 917-927；Feb.15,2012.).As needle in medical-therapeutic treatment of human body To the immune cell therapy drug of tumour, the antibody medicine for hindering the effect of LAG-3 is developed, clinical test (I phase) is being carried out (antibody name: BMS-986016, Bristol-Myers Squibb company and small open country pharmaceutical industries Zhu Shihui company).
Before this, the inventors of the present invention have carried out for animal refractory disease using LAG-3 as the immune cell therapy of target Exploitation, illustrate the novel immune cell therapy can across disease and across animal application (non-patent literature 4: Shirai T, Konnai S, Ikebuchi R, Okagawa T, Suzuki S, Sunden Y, Onuma M, Murata S, Ohashi K.Vet.Immunol.Immunopathol., 144 (3-4): 462-467；Dec.15,2011., non-patent literature 5: Konnai S, Suzuki S, Shirai T, Ikebuchi R, Okagawa T, Sunden Y, Mingala CN, Onuma M, Murata S, Ohashi K.Comp.Immunol.Microbiol.Infect.Dis., 36 (1): 63-69；Jan.2013., non- Patent document 6:Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R, Kawaji S, Tanaka S, Kagawa Y, Murata S, Mori Y, Ohashi K.Infect.Immun.84 (1): 77-89； Oct.19,2015.).
It, can not be to dynamic other than rat but since the inventors of the present invention's antibody made before this is rat Ab Object is applied repeatedly.
The prior art
Non-patent literature
Non-patent literature 1:Triebel F, Jitsukawa S, Baixeras E, Roman-Roman S, Genevee C, Viegas-Pequignot E, Hercend T.J.Exp.Med., 171 (5): 1393-1405；May 1,1990.
Non-patent literature 2:Blackburn SD, Shin H, HainingWN, Zou T, Workman CJ, Polley A, Betts MR, Freeman GJ, Vignali DA, Wherry EJ.Nat.Immunol., 10 (1): 29-37；Nov.30, 2008.
Non-patent literature 3:Woo S-R, Turnis ME, GoldbergM V., Bankoti J, Selby M, Nirschl CJ, Bettini ML, Gravano DM, Vogel P, Liu CL, Tangsombatvisit S, Grosso JF, Netto G, SmeltzerMP, Chaux A, Utz PJ, Workman CJ, Pardoll DM, Korman AJ, Drake CG, Vignali DAA.Cancer Res., 72 (4): 917-927；Feb.15,2012.
Non-patent literature 4:Shirai T, Konnai S, Ikebuchi R, Okagawa T, Suzuki S, Sunden Y, Onuma M, Murata S, Ohashi K.Vet.Immunol.Immunopathol., 144 (3-4): 462-467；Dec.15, 2011.
Non-patent literature 5:Konnai S, Suzuki S, Shirai T, Ikebuchi R, Okagawa T, Sunden Y, Mingala CN, Onuma M, Murata S, Ohashi K.Comp.Immunol.Microbiol.Infect.Dis., 36 (1): 63-69；Jan.2013.
Non-patent literature 6:Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R, Kawaji S, Tanaka S, Kagawa Y, Murata S, Mori Y, Ohashi K.Infect.Immun.84 (1): 77-89；Oct.19,2015.
Summary of the invention
Problem to be solved by the invention
It is anti-that the object of the present invention is to provide a kind of anti-lag-3s that the animal for other than rat can also apply repeatedly Body.
The method for solving problem
The inventors of the present invention have determined the anti-ox LAG-3 monoclonal antibody of rat with ox LAG-3 expression Cos-7 cell combination The variable region of (2D8), and by the variable region gene and cattle immune globulin (ox IgG1, wherein right in order to inhibit ADCC activity The Fc γ receptor anticipation binding site of CH2 structural domain is applied with mutation (referring to Fig.1.Amino acid serial number and mutation: 247E → P, 248L → V, 249P → A, 250G → missing, 344A → S, 345P → S；Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K.Immunology 2014Aug；142 (4): 551-561.).) constant region gene combination obtain chimeric antibody gene, culture proliferation has imported resulting chimeric antibody base Thus the Chinese hamster ovary cell (Chinese hamster ovary cell:CHO cell) of cause is successfully produced big Mouse-ox inosculating antibody ox LAG-3 antibody.Furthermore, it is determined that the CDR of the variable region of the anti-ox LAG-3 monoclonal antibody (2D8) of rat. The present invention is the invention completed according to these opinions.
Purport of the invention is as follows.
(1) a kind of anti-lag-3 antibody, it includes (a) L chain and (b) H chain, the L chain has comprising having The CDR1 of the amino acid sequence of QSLLDSDGNTY (sequence number 16), amino acid sequence with SVS CDR2 and have The L chain variable region of the CDR3 of the amino acid sequence of MQATHVPFT (sequence number 17) and the L chain of the animal's antibody other than rat are permanent Determine area；The H chain with the CDR1 comprising the amino acid sequence with GFDFDTYP (sequence number 18), have ITIKTHNYAT The H chain of the CDR3 of the CDR2 of the amino acid sequence of (sequence number 19) and the amino acid sequence with NREDFDY (sequence number 20) can Become the H chain constant region of the animal's antibody other than area and rat.
(2) according to the antibody recorded in (1), wherein L chain variable region and H chain variable region derive from rat.
(3) according to the antibody recorded in (2), wherein L chain variable region is the L chain variable region of the anti-ox LAG-3 antibody of rat, H Chain variable region is the H chain variable region of the anti-ox LAG-3 antibody of rat.
(4) according to the antibody recorded in (3), wherein L chain variable region has the amino acid sequence of sequence number 1, and H chain is variable Area has the amino acid sequence of sequence number 2.
(5) antibody recorded according to any one of (1)~(4), wherein the L chain constant region of the animal's antibody other than rat The amino acid sequence of constant region with Lambda chain or Kappa chain.
(6) the H chain constant region of the antibody recorded according to any one of (1)~(5), the animal's antibody other than rat has phase When the amino acid sequence or be imported into of the constant region of the immunoglobulin of the IgG4 in people keeps ADCC activity and/or CDC living Property reduce mutation.
(7) according to the antibody recorded in (6), wherein the animal other than rat is ox, and the L chain constant region of Niu Kangti has The amino acid sequence of the constant region of Lambda chain, and the H chain constant region of Niu Kangti has been imported into makes ADCC activity and/or CDC The mutation that activity reduces.
(8) according to the antibody recorded in (7), wherein the L chain constant region of Niu Kangti has the amino acid sequence of sequence number 3, The H chain constant region of ox antibody has the amino acid sequence of sequence number 4.
(9) antibody recorded according to any one of (1)~(8), four chain structures with two L chains and two H chains.
(10) a kind of medical composition, it includes the antibody of any one of (1)~(9) record as effective component.
(11) according to the medical composition recorded in (10), the prevention and/or treatment of cancer and/or infectious disease are used for.
(12) according to the medical composition recorded in (11), wherein cancer and/or infectious disease are selected from tumor disease, white Blood disease, chronic bacillary diarrhea, anaplasmosis (ア Na プ ラ ズ マ disease), bacillary mazoitis, fungoid mazoitis, mycoplasma infection (such as mycoplasma mazoitis, mycoplasmal pneumonia etc.), tuberculosis, small-sized piroplasmosis (small-sized ピ ロ プ ラ ズ マ disease), Cryptosporidiosis, globidiosis, trypanosomiasis and leishmaniasis.
(13) a kind of artificial gene DNA, it includes the DNA of (a ') coding L chain and the DNA, the L of (b ') coding H chain Chain is with the CDR1 comprising the amino acid sequence with QSLLDSDGNTY (sequence number 16), amino acid sequence with SVS The L chain variable region of the CDR3 of CDR2 and the amino acid sequence with MQATHVPFT (sequence number 17) and the animal other than rat resist The L chain constant region of body, the H chain with the CDR1 comprising the amino acid sequence with GFDFDTYP (sequence number 18), have The CDR2 of the amino acid sequence of ITIKTHNYAT (sequence number 19) and amino acid sequence with NREDFDY (sequence number 20) The H chain constant region of animal's antibody other than the H chain variable region of CDR3 and rat.
(14) a kind of carrier, it includes the artificial gene DNA recorded in (13).
(15) a kind of host cell is converted by the carrier recorded in (14).
(16) a kind of manufacturing method of antibody comprising the middle host cell recorded of culture (15) is simultaneously adopted from culture Collect the operation of anti-lag-3 antibody.
(17) a kind of DNA, encodes L chain, and the L chain is with including the amino with QSLLDSDGNTY (sequence number 16) The CDR1 of acid sequence, the CDR2 of amino acid sequence with SVS and the amino acid sequence with MQATHVPFT (sequence number 17) The L chain constant region of animal's antibody other than the L chain variable region of CDR3 and rat.
(18) a kind of DNA, encodes H chain, and the H chain is with including the amino acid sequence with GFDFDTYP (sequence number 18) The CDR1 of column, the CDR2 of amino acid sequence with ITIKTHNYAT (sequence number 19) and with NREDFDY (sequence number 20) The H chain constant region of animal's antibody other than the H chain variable region of the CDR3 of amino acid sequence and rat.
This specification includes the Japanese patent application Japanese Patent Application 2016-159091 on the basis of the priority as the application Specification and/or attached drawing in the content recorded.
Invention effect
According to the present invention it is possible to obtain novel anti-lag-3 antibody.The antibody can also benefit for the animal other than rat With.
Detailed description of the invention
Fig. 1 is rat-ox inosculating antibody ox LAG-3 antibody ch2D8 amino acid sequence.It is anti-to represent the anti-ox LAG-3 of rat The L chain variable region and the region CDR1, CDR2 and CDR3 in H chain variable region of body 2D8, in addition, being also illustrated with to ox IgG1 (CH2 Structural domain) be applied with mutation amino acid (amino acid serial number and mutation: 247E → P, 248L → V, 249P → A, 250G → lack It loses, 344A → S, 345P → S).
Fig. 2 is pDC6 carrier and rat-ox inosculating antibody ox LAG-3 antibody ch2D8 schematic diagram.
Fig. 3 is rat-ox inosculating antibody ox LAG-3 antibody ch2D8 purifying purity.
Fig. 4 is rat-ox inosculating antibody ox LAG-3 antibody ch2D8 associativity.
Fig. 5 is that rat-ox inosculating antibody ox LAG-3 antibody ch2D8 ox LAG-3/MHC II combines obstruction activity.
Fig. 6 is the variation of the response of the IFN-γ as caused by rat-ox inosculating antibody ox LAG-3 antibody ch2D8.
Fig. 7 is the cross reactivity of rat anti-ox LAG-3 antibody 2D8 and buffalo LAG-3.
Fig. 8 is the cross reactivity of the T cell of the anti-ox LAG-3 antibody 2D8 of rat and sheep.
Specific embodiment
Hereinafter, the present invention is described in detail.
The present invention provides a kind of anti-lag-3 antibody, and it includes (a) L chain and (b) H chain, the L chain has comprising having The CDR1 of the amino acid sequence of QSLLDSDGNTY (sequence number 16), amino acid sequence with SVS CDR2 and have The L chain variable region of the CDR3 of the amino acid sequence of MQATHVPFT (sequence number 17) and the L chain of the animal's antibody other than rat are permanent Determine area, the H chain with the CDR1 comprising the amino acid sequence with GFDFDTYP (sequence number 18), there is ITIKTHNYAT The H chain of the CDR3 of the CDR2 of the amino acid sequence of (sequence number 19) and the amino acid sequence with NREDFDY (sequence number 20) can Become the H chain constant region of the animal's antibody other than area and rat.
CDR1~3 in the L chain variable region of the anti-ox LAG-3 antibody 2D8 of rat are respectively comprising QSLLDSDGNTY (sequence Number 16) region of amino acid sequence, amino acid sequence comprising SVS region, include MQATHVPFT (sequence number 17) ammonia The region (referring to Fig.1) of base acid sequence.
In addition, CDR1~3 in the H chain variable region of the anti-ox LAG-3 antibody 2D8 of rat are respectively to include GFDFDTYP (sequence Row number 18) amino acid sequence region, comprising ITIKTHNYAT (sequence number 19) amino acid sequence region and comprising The region (referring to Fig.1) of the amino acid sequence of NREDFDY (sequence number 20).
In the amino acid sequence of QSLLDSDGNTY (sequence number 16), the amino acid sequence of SVS and MQATHVPFT (sequence number 17) amino of the amino acid sequence of amino acid sequence and GFDFDTYP (sequence number 18), ITIKTHNYAT (sequence number 19) In the amino acid sequence of acid sequence and NREDFDY (sequence number 20), 1,2,3,4 or 5 can be lacked, replaces or is added A amino acid, even if importing these mutation, it is possible to have CDR the or H chain variable region of the L chain variable region as LAG-3 antibody CDR function.
In this specification, so-called antibody is other than including full length antibody, further includes Fab, F (ab) '2, ScFv, two Poly- antibody, VH、VL、Sc(Fv)2, bispecific sc (Fv)2, miniantibody, ScFv-Fc monomer, ScFv-Fc dimer etc. it is low The concept of the antibody of molecularization.
In anti-lag-3 antibody of the invention, L chain variable region and H chain variable region can derive from rat.For example, it may be L Chain variable region is the L chain variable region of the anti-ox LAG-3 antibody of rat, and H chain variable region is that the H chain of the anti-ox LAG-3 antibody of rat is variable Area.
The amino acid sequence of the L chain variable region of the anti-ox LAG-3 antibody of rat and the amino acid sequence of H chain variable region are distinguished It is shown in sequence number 1 and 2, however in the amino acid sequence of sequence number 1 and 2, it can lack, replace or be added 1 or multiple (such as 5 or less, at most 10 or so) amino acid, even if importing these mutation, it is possible to have the L as LAG-3 antibody The function of chain variable region or H chain variable region.
In the L chain of antibody, there are Kappa chain (Kappa chain) and Lambda chain (lambda chain), anti-lag-3 of the invention is anti- In body, no matter the L chain constant region of the antibody of the animal other than rat has the constant region of which chain of Kappa chain or Lambda chain Amino acid sequence be ok, for there are ratios, as soon as with regard to ox, sheep, cat, dog, Ma Eryan be Lambda chain side it is high, it is small It is the side height of Kappa chain for mouse, rat, people, pig.Due to the side for thinking preferably to have the high chain of ratio, with regard to ox, Preferably there is the amino acid sequence of the constant region of Lambda chain for sheep, cat, dog, horse, it is excellent for mouse, rat, people, pig Select the amino acid sequence with the constant region of Kappa chain (Kappa chain).
The H chain constant region of the antibody of animal other than rat can have the perseverance for being equivalent to the immunoglobulin of IgG4 of people Determine the amino acid sequence in area.H chain is divided into γ chain, μ chain, α chain, δ chain, ε chain according to the difference of constant region, is distinguished according to the difference Form the immunoglobulin of 5 seed types (isotype) of IgG, IgM, IgA, IgD, IgE.
Immunoglobulin G (IgG) accounts for the 70-75% of human immunoglobulin(HIg), is the antibody of monomer most in blood plasma.Have Four chain structures of two light chains and two heavy chains.Human IgG1, IgG2, IgG4 molecular weight be about 146000, and the company of human IgG 3 The hinge minister in the region Fab Yu the region Fc is met, molecular weight is also greatly to 170000.Human IgG1 accounts for 65% of human IgG or so, human IgG2 25% or so is accounted for, human IgG 3 accounts for 7% or so, and human IgG 4 accounts for 3% or so.It is evenly distributed within extra vascular.Human IgG1 is due to right Fc receptor, the complement factor on effector cell surface have strong compatibility, therefore induction of antibodies dependent cell toxic action (ADCC), In addition, the inducing complement dependent cell toxic action (CDC) by complement activation.Human IgG2 and human IgG 4 are due to Fc receptor, mending The compatibility of the body factor is low, therefore ADCC activity and CDC activity are low.
Immunoglobulin M (IgM) be account for human immunoglobulin(HIg) about 10%, obtain in conjunction with 5 four basic chain structures Pentamer antibody.Molecular weight is 970000.It is usually only present in blood, generated, served as at first for microbial infection The immunoglobulin of early immune.
Immunoglobulin A (IgA) accounts for the 10-15% of human immunoglobulin(HIg).Molecular weight is 160000.Secretory IgA is knot Close the antibody of dimer obtained by 2 IgA.IgA1 is present in serum, nasal mucus, saliva, in breast milk, exists in intestinal juice a large amount of IgA2。
Immunoglobulin D (IgD) is the antibody of 1% monomer below of human immunoglobulin(HIg).It is present in B cell surface, Participate in the induction that antibody generates.
Immunoglobulin E (IgE) is the anti-of monomer existing for 0.001% only denier below of human immunoglobulin(HIg) Body.It is considered the immune response for participating in being directed to helminth, and in the developed country of helminth rareness, especially roar with bronchus Asthma, allergy have much relations.
For dog, as the H chain of IgG, identify IgG-A (being equivalent to human IgG2), IgG-B (being equivalent to human IgG1), The sequence of IgG-C (being equivalent to human IgG 3), IgG-D (being equivalent to human IgG 4).In antibody of the invention, preferably do not have simultaneously The active IgGH chain constant region of ADCC activity, CDC (ratione personae is IgG4).The immune of human IgG 4 is equivalent to not identifying In the case where the constant region of globulin, it can be used and mutation is applied by the region to the immunoglobulin for being equivalent to human IgG1 Without having the active region of ADCC activity, CDC simultaneously.
The sequence of IgG1, IgG2, IgG3 are identified as the H chain of IgG with regard to Niu Eryan.In antibody of the invention, preferably There is no the active IgGH chain constant region of ADCC activity, CDC simultaneously (ratione personae is IgG4).The constant region of known human IgG1 is just There is ADCC activity and CDC activity for wild type, and by applying amino acid replacement, missing to specific part, it can make Their activity reduces.In ox, the constant region of the immunoglobulin of human IgG 4 is equivalent to due to not identifying, it can be with The region for the immunoglobulin for being equivalent to human IgG1 is applied and is mutated and uses the region.It, will be to ox antibody as its an example The CH2 structural domain of H chain constant region (IgG1 chain, GenBank:X62916) be applied with the amino acid sequence and nucleotides sequence of mutation Column (by codon-optimized sequence) respectively indicate in sequence number 4 and 8.
More preferably following anti-lag-3 antibody, that is, the L chain constant region of Niu Kangti has the ammonia of the constant region of Lambda chain Base acid sequence, and the H chain constant region of Niu Kangti has been imported into the mutation for reducing ADCC activity and/or CDC activity.
Anti-lag-3 antibody of the invention includes rat-ox chimeric antibody, oxization antibody, complete ox type antibody, however dynamic Object is not limited to ox, may be exemplified out people, dog, pig, monkey, mouse, cat, horse, goat, sheep, buffalo, rabbit, hamster, cavy etc. Deng.
For example, the L chain constant region that anti-lag-3 antibody of the invention can be Niu Kangti has the amino acid sequence of sequence number 3 It arranges, the anti-lag-3 antibody of amino acid sequence of the H chain constant region of Niu Kangti with sequence number 4.
In the amino acid sequence of sequence number 3 and 4, can lack, replace or be added 1 or multiple (such as 5 or less, extremely More 10 or so) amino acid, even if importing these mutation, it is possible to have L chain constant region or H chain constant region as antibody Function.
Anti-lag-3 antibody of the invention can be four chain structures with two L chains and two H chains.
Anti-lag-3 antibody of the invention can manufacture as shown below.Synthesis is anti-comprising the anti-ox LAG-3 of rat identified The variable region sequences of body and the antibody of the animal (such as ox etc.) other than rat are (preferably to the immune globulin for being equivalent to human IgG1 The antibody that the white region applies mutation, reduces ADCC activity and/or CDC activity) constant-region sequences artificial gene, will It after carrier (such as plasmid) is added in the artificial gene, imports host cell (such as the mammalian cells such as Chinese hamster ovary celI), and cultivating should Thus host cell acquires antibody from culture.
The amino acid sequence and nucleotide of the L chain variable region of the anti-ox LAG-3 antibody of the rat that the inventors of the present invention are identified Sequence respectively indicates in sequence number 1 and 5.In addition, the nucleotide sequence after will be codon-optimized is shown in sequence number 11.
The amino acid sequence and nucleotide of the H chain variable region of the anti-ox LAG-3 antibody of the rat that the inventors of the present invention are identified Sequence respectively indicates in sequence number 2 and 6.In addition, the nucleotide sequence after will be codon-optimized is shown in sequence number 12.
By the amino acid sequence and nucleotide sequence of the L chain constant region (Lambda chain, GenBank:X62917) of Niu Kangti It respectively indicates in sequence number 3 and 7.In addition, the nucleotide sequence after will be codon-optimized is shown in sequence number 13.
By the amino acid sequence and nucleotides sequence of the H chain constant region (IgG1 chain changes GenBank:X62916) of Niu Kangti Column (after codon-optimized) respectively indicate in sequence number 4 and 8.
In addition, sequence number 9 indicates the L chain variable region comprising the anti-ox LAG-3 antibody of rat and the L chain constant region of Niu Kangti The amino acid sequence of the chimeric L chain of (Lambda chain, GenBank:X62917).It can by the L chain comprising the anti-ox LAG-3 antibody of rat Become the nucleotide sequence (codon of the chimeric L chain of area and the L chain constant region (Lambda chain, GenBank:X62917) of Niu Kangti After optimization) it is shown in sequence number 14.
Sequence number 10 indicates the H chain variable region comprising the anti-ox LAG-3 antibody of rat and the H chain constant region (IgG1 of Niu Kangti Chain, change GenBank:X62916) chimeric H chain amino acid sequence.H chain comprising the anti-ox LAG-3 antibody of rat can be changed Nucleotide sequence (the codon of the chimeric H chain of the H chain constant region (IgG1 chain changes GenBank:X62916) of area and Niu Kangti After optimization) it is shown in sequence number 15.
In addition to this, the amino acid sequence and nucleotide sequence of the L chain constant region of the animal other than rat and H chain constant region It can be obtained from well known database, can use these sequences.
The amino acid sequence and nucleotide sequence of the L chain constant region of ox and H chain constant region are concentrated and arranged in following tables In.
(table)
The amino acid sequence and nucleotide sequence of sheep, buffalo, the L chain constant region of people and H chain constant region are concentrated and arranged In following tables.
(table)
In the amino acid sequence of sequence number 3,21~28,37,39,41,43,45,47,49,51,53,55,57 and 59 In, 1 or multiple (such as 5 or less, at most 10 or so) amino acid can be lacked, replaces or be added, even if it is prominent to import these Become, it is possible to have the function of the constant region as Ig heavy chain or light chain.
The constant region of known human IgG1 has ADCC activity and CDC activity for wild type, however by specific Part applies amino acid replacement, missing, can reduce their activity.In the animal other than people, do not identifying quite In the case where the constant region of the immunoglobulin of human IgG 4, the area to the immunoglobulin for being equivalent to human IgG1 can be used Domain applies mutation, reduces ADCC activity and the active region CDC.
The present invention provides a kind of artificial gene DNA, and it includes the DNA of the DNA of (a ') coding L chain and (b ') coding H chain, institutes State L chain with the CDR1 comprising the amino acid sequence with QSLLDSDGNTY (sequence number 16), with the amino acid sequence of SVS CDR2 and with MQATHVPFT (sequence number 17) amino acid sequence CDR3 L chain variable region and the animal other than rat The L chain constant region of antibody, the H chain with the CDR1 comprising the amino acid sequence with GFDFDTYP (sequence number 18), have The CDR2 of the amino acid sequence of ITIKTHNYAT (sequence number 19) and amino acid sequence with NREDFDY (sequence number 20) The H chain constant region of animal's antibody other than the H chain variable region of CDR3 and rat.In addition, the present application also provides a kind of coding The DNA of L chain, the L chain with the CDR1 comprising the amino acid sequence with QSLLDSDGNTY (sequence number 16), with SVS's The L chain variable region of the CDR3 of the CDR2 of amino acid sequence and the amino acid sequence with MQATHVPFT (sequence number 17) and rat The L chain constant region of animal's antibody in addition.In addition, the present invention also provides a kind of DNA for encoding H chain, the H chain has comprising tool CDR1, the amino acid sequence with ITIKTHNYAT (sequence number 19) for having the amino acid sequence of GFDFDTYP (sequence number 18) The H chain variable region of the CDR3 of CDR2 and the amino acid sequence with NREDFDY (sequence number 20) and the animal other than rat resist The H chain constant region of body.
For (a) with the CDR1 comprising the amino acid sequence with QSLLDSDGNTY (sequence number 16), with SVS's The L chain variable region of the CDR3 of the CDR2 of amino acid sequence and the amino acid sequence with MQATHVPFT (sequence number 17) and rat The L chain constant region L chain of animal's antibody in addition and (b) with including the amino acid sequence with GFDFDTYP (sequence number 18) The CDR1 of column, the CDR2 of amino acid sequence with ITIKTHNYAT (sequence number 19) and with NREDFDY (sequence number 20) The H chain of the H chain constant region of animal's antibody other than the H chain variable region of the CDR3 of amino acid sequence and rat, is chatted in front It states.The DNA of (a ') is the DNA (gene) for encoding the L chain of (a), and the DNA of (b ') is the DNA (gene) for encoding the H chain of (b), includes Commercially available synthesizer synthesis can be used in the artificial gene DNA of the DNA of the DNA of (a ') and (b ').It can also be to artificial gene DNA Restriction enzyme recognition site, KOZAK sequence, poly (A) tailing signal sequence, promoter sequence, intron sequences etc. are added.
In addition, the present invention also provides the carriers comprising the artificial gene DNA.
As carrier, the plasmid (such as pBR322, pBR325, pUC12, pUC13), withered of Escherichia coli can be used Plasmid (such as pUB110, pTP5, pC194), the yeast sources plasmid (such as pSH19, pSH15), λ bacteriophage in straw bacterium source Insect Pathogenics viruses such as animal virus, the baculovirals such as equal bacteriophages, retrovirus, vaccinia virus etc..Aftermentioned embodiment In, use pDC6 (Japanese Patent No. 5704753, US patent 9096878, EU patent 2385115, Hong Kong (China) patent HK1163739, Australian Patent 2009331326).
Can also be added into carrier promoter, enhancer, splicing signal, poly (A) tailing signal, intron sequences, Selected marker, SV40 replication origin etc..
In addition, the present invention also provides the host cells converted using the carrier.By cultivating the host cell, And antibody is acquired from culture, anti-lag-3 antibody can be manufactured.As a result, the present invention also provides a kind of manufacturing method of antibody, The manufacturing method includes the operation cultivated the host cell and acquire anti-lag-3 antibody from culture.Antibody of the invention Manufacturing method in, can will incorporate comprising coding L chain DNA and coding H chain DNA artificial gene DNA carrier turn It contaminates in host cell, it can also be by the carrier corotation of the carrier for incorporating the DNA of coding L chain and the DNA for incorporating coding H chain It contaminates in host cell.
As host cell, bacterial cell (such as Escherichia bacterium, bacillus, withered grass may be exemplified out Bacillus etc.), fungal cell's (such as yeast, aspergillus etc.), insect cell (such as S2 cell, Sf cell etc.), zooblast (such as Chinese hamster ovary celI, COS cell, HeLa cell, C127 cell, 3T3 cell, bhk cell, HEK293 cell etc.), plant cell etc..Its In, preferably as the CHO-DG44 cell (CHO-DG44 (dhfr-/-) of dihyrofolate reductase defect cell).
Recombinant vector is imported into host, can use the molecular cloning second edition, J.Sambrook et al., Cold SpringHarborLab.Press, the method recorded in 1989 (such as it is calcium phosphate method, DEAE- glucan method, infection protocol, micro- Injection method, lipofection, electroporation, conversion method, scraper load method (ス ク レ ー プ ロ ー デ ィ Application グ method), air gun Method (シ ョ ッ ト ガ Application method) etc.) or infect to carry out.
Culture medium culture transformant can be used, anti-lag-3 antibody of the invention is acquired from culture.It is secreted in antibody In the case where into culture medium, as long as recycling culture medium and separation, antibody purification from the culture medium.It is resulted from antibody By convert it is intracellular in the case where, as long as by the cell dissolution and from its dissolved matter separation, antibody purification.
As culture medium, OptiCHO culture medium, Dynamis culture medium, CD CHO culture medium, ActiCHO may be exemplified out Culture medium, FortiCHO culture medium, Ex-Cell CD CHO culture medium, BalanCD CHO culture medium, 5 culture medium of ProCHO, Cellvento CHO-100 culture medium etc., however it is not limited to them.
The pH of culture medium is different according to the cell cultivated, however is in general pH6.8~7.6, in most cases It is suitably for pH7.0~7.4.
In the case where the cell cultivated is Chinese hamster ovary celI, the culture of Chinese hamster ovary celI be can be used well known to those skilled in the art Method carries out.For example, usually can be in the CO of gas phase2Concentration be 0-40%, preferably 2-10% atmosphere under, in 30-39 DEG C, preferably cultivated at 37 DEG C or so.
It is usually 1 day~3 months, preferably 1 day~3 weeks during suitable culture.
The separation and purifying of antibody can use well known method and carry out.As well known separation, method of purification, can be used Saltout, solvent precipitation etc. using the difference of solubility method, dialysis, ultrafiltration, gel filtration and SDS- poly- third Acrylamide gel electrophoresis etc. using molecular weight method, ion-exchange chromatography of difference etc. using charge difference method, affine Chromatography etc. is electric using method, the isoelectric point of hydrophobic difference using method, reverse phase high-speed liquid chromatography of special sexual compatibility etc. Swimming method etc. utilizes the method etc. of the difference of isoelectric point.
Anti-lag-3 antibody of the invention can be used as animal with or people antibody medicine utilize.The present invention provides packet as a result, Medical composition containing above-mentioned anti-lag-3 antibody as effective component.
Medical composition of the invention can be used for the prevention and/or treatment of cancer and/or infectious disease.As cancer and/ Or infectious disease, may be exemplified out tumor disease (such as malignant mela noma, lung cancer, gastric cancer, kidney, breast cancer, bladder cancer, Cancer of the esophagus, Luan Testis cancer etc.), leukaemia, chronic bacillary diarrhea, anaplasmosis, bacillary mazoitis, fungoid mazoitis, mycoplasma sense Contaminate disease (such as mycoplasma mazoitis, mycoplasmal pneumonia etc.), tuberculosis, small-sized piroplasmosis, Cryptosporidiosis, coccidia Disease, trypanosomiasis and leishmaniasis etc., however it is not limited to them.
Anti-lag-3 antibody of the invention can be dissolved in the buffers such as PBS, physiological saline, aqua sterilisa etc., according to need After being filtered sterilizing with filter etc., applied using injection to animal subject (also including people).Furthermore it is possible to the solution Middle addition additive (such as it is colorant, emulsifier, suspending agent, surfactant, dissolution aids, stabilization agent, preservative agent, anti- Oxygen agent, buffer, isotonic agent, pH adjusting agent etc.) etc..As administration route, vein, muscle, abdominal cavity, subcutaneous, intradermal can be Application etc., alternatively, it is also possible to intranasal, oral administration.
The amount of application of anti-lag-3 antibody, the number of application and frequency of the invention according to the symptom of animal subject, the age, Weight, method of administration, application form etc. and it is different, such as usually can to each adults at least once, can obtain The frequency of desired effect applies 0.1~100mg/kg weight, preferably 1~10mg/kg weight.
Medical composition of the invention can be used alone, other can also exempt from surgical operation, radiotherapy, cancer vaccine etc. Epidemic disease cell therapy, molecular targeted therapy medicine are applied in combination.It is possible thereby to expect synergistic effect.
Embodiment
Hereinafter, based on embodiment, the present invention is described in detail, however the present invention is not limited to these embodiments.
The foundation of (embodiment 1) rat-ox inosculating antibody ox LAG-3 antibody
1. introduction
Immunosupress receptor lymphocyte activationgene 3 (LAG-3) is accredited as nearly fate of CD4, It illustrates in recent years and participates in chronic infection disease, the immunosupress in tumour.In the present embodiment, to establish to the novel of the infectious disease of ox For the purpose of cure, rat-ox inosculating antibody ox LAG-3 antibody ch2D8 has been made, and confirmed to be combined by this antibody bring Activity and bioactivity are hindered, the rat-ox inosculating antibody ox LAG-3 antibody ch2D8 is that culture proliferation has imported chimeric antibody The Chinese hamster ovary cell (Chinese hamsterovary cell:CHO cell) of gene and obtain, the chimeric antibody base Cause is the variable region of the anti-ox LAG-3 monoclonal antibody 2D8 of rat for the combination that will hinder ox LAG-3 and MHC class II Gene and cattle immune globulin (ox IgG1 and Ig λ.Wherein, in order to inhibit ADCC activity, to the Fc γ of ox IgG1CH2 structural domain Receptor anticipation binding site is applied with mutation (Fig. 1).Amino acid serial number and mutation: 247E → P, 248L → V, 249P → A, 250G → missing, 344A → S, 345P → S；Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K.Immunology, 142 (4): 551-561；Aug.2014. constant region gene group) It closes and obtains.
2. material, method and experimental result
2.1. the building of ox LAG-3 expression cell
To ox LAG-3 gene (GenBank registration number AB608099；Shirai T, Konnai S, Ikebuchi R, Okagawa T, Sunden Y, Onuma M, Murata S, Ohashi K.Vet.Immunol.Immunopathol, 144 (3- 4): 462-467；Dec.15,2011.) base sequence for determining cDNA overall length, produces ox LAG-3 table according to its gene information Up to cell.Firstly, in order to make ox LAG-3 expression plasmid, with synthesized ox peripheral blood mononuclear cells (PBMC) source cDNA For template, using be added to 5 ' end sides restriction enzyme BglII and EcoRI recognition site primer (boLAG-3-EGFP F and R PCR) has been carried out.Resulting PCR product is handled using BglII (Takara company) and EcoRI (Takara company) Afterwards, it is purified using Fast Gene gel/PCR extracts kit (NIPPON Genetics company), importing has carried out same In the pEGFP-N2 carrier (Clontech company) of the restriction enzyme processing of sample, cloned.Use QIAGEN Plasmid Midi kit (Qiagen company) extracts the expression plasmid of resulting target, for being saved before testing with -30 DEG C.After, it will Made expression plasmid is expressed as pEGFP-N2-boLAG-3.
Primer (boLAG-3-EGFP F):
GAAAGATCTATGCTGTGGGAGGCTTGGTT (sequence number 61)；
Primer (boLAG-3-EGFP R):
CCGGAATTCGGGTTGCTCTGGCTGCAGCT (sequence number 62).
According to following step, ox LAG-3 expression cell is produced.Firstly, by 5 × 104A/cm2COS-7 cell 6 It is passed in orifice plate, is including 10% passivation fetal calf serum (Cell Culture Technologies company), penicillin 200U/ml, 200 μ g/ml of streptomysin, 0.01%L- glutamic acid (Life Technologies company) 1640 culture medium of RPMI In 37 DEG C, 5%CO in (Sigma-Aldrich company)2Under the conditions of cultivate a Dinner.Then, using Lipofectamine 2000 (Invitrogen company) imports 0.4 μ g/cm into COS-7 cell2PEGFP-N2-boLAG-3 or as negative control PEGFP-N2 and cultivate 48 hours (ox LAG-3-EGFP expression cell).In order to confirm the ox in made expression cell The expression of LAG-3 makes the intracellular part of EGFP can using inverted type confocal laser microscope LSM700 (ZEISS company) Depending on changing.
2.2. the building of soluble ox LAG-3
According to following step, ox LAG-3-Ig expression plasmid is constructed.To expand ox LAG-3 (GenBank registration number AB608099 the mode of signal peptide and extracellular space) devises to 5 ' end sides and restriction enzyme NheI and NsiI knowledge is added The primer (boLAG-3-Ig F and R) at other position.PCR is carried out using synthesized ox PBMC source cDNA out as template, PCR is produced After object is handled using NheI (Takara company) and NsiI (Takara company), is extracted and tried using FastGene Gel/PCR Agent box (NIPPON Genetics company) is purified, and the pCXN2.1- for having carried out same restriction enzyme processing is imported Rabbit IgG1Fc carrier (Niwa H, Yamamura K, Miyazaki J.gene, 108 (2): 193-199；Dec.15, 1991；Change by Juntendo University big institute's medical research section traverse furrow professor Yue Yan point and carrier), cloned.By table It is purified up to plasmid using FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics company), for real It is saved before testing with -30 DEG C.After, made expression plasmid is expressed as pCXN2.1-boLAG-3-Ig.
Primer (boLAG-3-Ig F):
CTAGCTAGCCGCCCACCATGCTGTGGGAGGCTTGGTT (sequence number 63)；
Primer (boLAG-3-Ig R): TGCATGCATCAGAACAGCTAGGTTGTACG (sequence number 64).
According to following step, soluble ox LAG-3-Ig expression cell is produced.To 7.5 × 107A Expi293F 30 μ g are imported using Expifectamine (Life Technologies company) in cell (Life Technologies company) PCXN2.1-boLAG-3-Ig, carry out recycling culture supernatant after shaken cultivation in 7 days.Ab- is used from culture supernatant Capcher Extra (ProteNova company) purifies recombinant protein.After purification, PD MiniTrap G-25 (GE is utilized Healthcare company) by buffer exchange be PBS (pH 7.4), for before testing with -30 DEG C of preservations (ox LAG-3-Ig). The dense of ox LAG-3-Ig after purification is determined using Rabbit IgG ELISA Quantitation Set (Bethyl company) Degree.Using automatic plate washer BIO WASHER 50, (DS Pharma Biomedical is public in each cleaning operation of ELISA Department), microplate reader MTP-650FA (electric corporation CORONA) is used in the measurement of absorbance.
2.3. the anti-ox LAG-3 monoclonal antibody of rat produces celliferous production
Synthesize a part (amino acid serial number 71~99 in the extracellular space of ox LAG-3；Amino acid sequence: GSAAPTPRGPGPRRYTVLRLAPGGLRIGK (sequence number 72)) to NH2End joined the peptide chain of cysteine residues, with Keyhole limpet hemocyanin (keyhole limpet hemocyanin) as carrier protein combines.Make the peptide chain with The lotion of TiterMax Gold Adjuvant (Sigma-Aldrich company), is immunized the vola pedis of rat.Thereafter, Hybridoma is established using iliacal lymph nodes method, the anti-ox LAG-3 monoclonal antibody of rat is obtained and generates hybridoma 2D8 plants.For big The method for building up of the anti-ox LAG-3 monoclonal antibody of mouse, recorded in non-patent literature below its details (Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R, Kawaji S, Tanaka S, Kagawa Y, Murata S, Mori Y, Ohashi K.Infect.Immun., 84 (1): 77-89；Oct.19,2015.).
2.4. rat-ox inosculating antibody ox LAG-3 antibody expression vector production
Establish the antibody constant that ox IgG1 and ox Ig λ have been merged using the anti-ox LAG-3 antibody 2D8 of rat as antibody variable region The rat in area-ox inosculating antibody ox LAG-3 antibody ch2D8.
Firstly, identifying variable region (heavy chain using RACE method from the hybridoma for generating the anti-ox LAG-3 antibody 2D8 of rat And light chain) gene.Then, the heavy chain for making the anti-ox LAG-3 antibody 2D8 of rat and light-chain variable sequence and known ox is made IgG1 (heavy chain；Change GenBank registration number X62916) and ox Ig λ (light chain；GenBank registration number X62917) constant region knot The gene order of conjunction, has carried out that codon-optimized ((codon is most for sequence number 9 and 10 (amino acid sequences), sequence number 14 and 15 Nucleotide sequence after goodization)).It should be noted that in order to inhibit ADCC activity in ox IgG1, to the Fc γ of CH2 structural domain by Body anticipation binding site is applied with mutation (referring to Fig.1.Amino acid serial number and mutation: 247E → P, 248L → V, 249P → A, 250G → missing, 344A → S, 345P → S；Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K.Immunology, 142 (4): 551-561；Aug.2014.).Then, will NotI restricted enzyme recognition sequence, chimeric antibody light sequence, poly (A) tailing signal sequence (PABGH), opens KOZAK sequence Promoter sequences (PCMV), the restricted enzyme recognition sequence of SacI, intron sequences (INRBG), KOZAK sequence, chimeric antibody heavy The restricted enzyme recognition sequence of sequence, XbaI artificially synthesizes gene according to the above-mentioned mode being arranged in order.It will be synthesized After gene strand is using NotI (Takara company) and XbaI (Takara company) processing, is extracted and tried using FastGene Gel/PCR Agent box (NIPPON Genetics company) purifying imports the expression plasmid pDC6 for having carried out same restriction enzyme processing (by north Seaway university infecting both domestic animals and human infectious disease research center Suzuki determine a professor man of virtue and ability point with) cloning site (positioned at the downstream PCMV, INRBG The restricted enzyme recognition sequence of NotI and XbaI between PABGH), cloned (Fig. 2).Use QIAGEN Plasmid Midi kit (Qiagen company) extracts the expression plasmid of resulting target, for being saved before testing with -30 DEG C.After, it will Made expression plasmid is expressed as pDC6-boLAG-3ch2D8.
2.5. rat-ox inosculating antibody ox LAG-3 antibody expression
Produced pDC6-boLAG-3ch2D8 is used into Lipofectamine LTX (Life technologies Company) it imports as dihyrofolate reductase defect (dfhr-/-) cell CHO-DG44 cell.After 48 hours, more by culture medium It is changed to the CD OptiCHO culture medium (Life comprising 2mM GlutaMAX additive (Life technologies company) Technologies company), it cultivates 3 weeks and has carried out the selection based on expression cell and the clone of limiting dilution method.Then, sharp With in dot blotting and ELISA method the measurement culture for having used anti-ox IgG F (c) rabbit polyclonal antibody (Rockland company) The concentration of chimeric antibody contained in clear liquid, filters out high-expression clone.By the rat established as described above-ox inosculating antibody ox LAG-3 antibody stabilization expression cell is transferred in CD OptiCHO culture medium, carry out 14 days shaken cultivation (125rpm, 37 DEG C, 5%CO2).Using the ELISA method for having used anti-ox IgG F (c) rabbit polyclonal antibody (Rockland company), to culture supernatant Chimeric antibody yield in liquid is quantified.It should be noted that having used automatic washing in each cleaning operation of ELISA Trigger BIO WASHER 50 (DS Pharma Biomedical company), has used microplate reader in the measurement of absorbance MTP-650FA (electric corporation CORONA).By the 14th day culture supernatant with 10000g centrifugation 10 minutes and after removing cell, Centrifuged supernatant is sterilized by 0.22 μm of filter of Steritop-GP (Millipore company), for before purification with 4 DEG C save.
2.6. rat-ox inosculating antibody ox LAG-3 antibody purifying
From the culture supernatant that benefit prepares with the aforedescribed process, using Ab Capcher Extra, (ProteNova is public Department) each chimeric antibody is purified.Open tubular column method is used in combination with resin, uses as equilibrating buffer and cleaning buffer solution 1.5M Glycine/3M NaCl(pH 8.0).0.1M Glycine-HCl (pH 2.8) is used as elution buffer, as Neutralization buffer uses 1M Tris (pH 9.0).To sublimed antibody, using PD-10 desalting column, (GE Healthcare is public Department) and Amicon Ultra-15 (50kDa, Millipore company) carried out the buffer exchange for being replaced into PBS (pH 7.4) And concentration.Sublimed chimeric antibody is carried out by 0.22 μm of syringe filter (Pall Life Sciences company) Sterilizing, for being saved before testing with 4 DEG C.
2.7. the confirmation (Fig. 3) of rat-ox inosculating antibody ox LAG-3 antibody purifying purity
In order to confirm sublimed rat-ox inosculating antibody ox LAG-3 antibody purity, dyed using SDS-PAGE and CBB The detection of antibody protein is carried out.Sublimed rat-ox inosculating antibody ox LAG-3 antibody ch2D8 is suspended in Laemmli In sample buffer (Bio-Rad company), (restored under the reducing conditions using 2 mercapto ethanol (Sigma-Aldrich company)) Or denaturation treatment (95 DEG C, 5 minutes) are carried out under non reducing conditions.To prepared sample using 10% polyacrylamide gel into Row electrophoresis.At this point, having used the full blue protein standard items (Bio- of Precision Plus Protein as molecular weight marker Rad company).After electrophoresis, the dyeing of gel is carried out using Quick-CBB (and Wako Pure Chemical Industries company), next in distilled water In decolourize.
It the results are shown in Fig. 3.Under the reducing conditions in the position of 25kDa (light chain) and 50kDa (heavy chain) estimated Rat-ox inosculating antibody ox LAG-3 antibody band is confirmed, in the position of 150~250kDa estimated under non reducing conditions Confirm rat-ox inosculating antibody ox LAG-3 antibody band.
2.8. rat-ox inosculating antibody ox LAG-3 antibody binding specificity (Fig. 4)
It confirmed that rat-ox inosculating antibody ox LAG-3 antibody and ox LAG-3 expression cell are (preceding using Flow cytometry State) specifically combine.Firstly, make the anti-ox LAG-3 antibody 2D8 of rat or rat-ox inosculating antibody ox LAG-3 antibody ch2D8 with Ox LAG-3 expression cell is reacted 30 minutes at room temperature.After cleaning, allophycocyanin (APC) is made to mark anti-rat Ig goat anti- Body (SouthernBiotech company) or Alexa Fluor 647 mark anti-ox IgG (H+L) goat F (ab')2(Jackson ImmunoResearch company) it reacts 30 minutes at room temperature.As negative control antibody, use rat IgG1 (κ) of the same race Type compares (BD Biosciences company) or ox IgG1 antibody (Bethyl company).After cleaning, FACS Verse (BD is utilized Biosciences company) detect each rat Ab for being incorporated into cell surface or rat-ox chimeric antibody.It needs to illustrate It is that in the dilution of all cleaning operation and antibody, having used addition to have 1% bovine serum albumin(BSA), (Sigma-Aldrich is public Department) PBS.
Experimental result is shown in Fig. 4.Show rat-ox inosculating antibody ox LAG-3 antibody ch2D8 and the anti-ox of rat LAG-3 antibody 2D8 is similarly in conjunction with ox LAG-3 expression cell.
2.9. rat-ox inosculating antibody LAG-3 antibody ox LAG-3/MHC class II, which is combined, hinders active (Fig. 5)
Use (the ox B cell lymthoma derived cell strain of BL3.1 cell strain；Strongly expressed MHC class II) and ox LAG- 3-Ig (aforementioned) combined using the ox LAG-3/MHC class II of anti-lag-3 antibody and has been hindered test.Firstly, 96 By the anti-ox LAG-3 antibody 2D8 of rat of final concentration (0,1.56,3.12,6.25,12.5,25, μ g/ml) or rat-in orifice plate Ox inosculating antibody ox LAG-3 antibody ch2D8 is mixed with the ox LAG-3-Ig of 3.3 μ g/ml of final concentration, is reacted 30 minutes at 37 DEG C.So Afterwards, by 1 × 105A BL3.1 cell strain has 10% passivation lowlenthal serum (Life Technologies company) using addition PBS reacts 30 minutes with above-mentioned mixed liquor at 37 DEG C after room temperature is closed 15 minutes.As negative control antibody, use Rat IgG1 (κ) isotype controls (BD Biosciences company) or ox IgG1 antibody (Bethyl company).After cleaning, make thing First implemented with rat blood serum source IgG (Sigma-Aldrich company) and derived therefrom IgG (Sigma-Aldrich company) The Alexa Fluor 647 of absorption processing (37 DEG C, 30 minutes) marks anti-rabbit IgG (H+L) goat F (ab')2(Life Technologies company) it reacts 30 minutes at room temperature, detect the ox LAG-3-Ig for being incorporated into cell surface.Make when analysis With FACS Verse (BD Biosciences company).It should be noted that in the dilution of all cleaning operation and antibody It is middle to have used the PBS for being added and having 1% bovine serum albumin(BSA) (Sigma-Aldrich company).Ox LAG- when will be not added with antibody The ratio of 3-Ig combination cell is set as 100%, and the ox LAG-3-Ig combination cell under each antibody concentration is represented as relative value Ratio.
Experimental result is shown in Fig. 5.Rat-ox inosculating antibody ox LAG-3 chimeric antibody ch2D8 with the anti-ox of rat The identical degree of LAG-3 antibody 2D8 hinders the combination of LAG-3-Ig and LAG-3 expression cell.
2.10. rat-ox inosculating antibody ox LAG-3 antibody biological activity test (Fig. 6) has been used in order to confirm by big Ox LAG-3/MHC class II caused by mouse-ox inosculating antibody ox LAG-3 antibody, which is combined, to be hindered lymphocyte activation, with IFN-γ yield has carried out biological activity test as index.By the PBMC isolated from the peripheral blood of ox with reach 2 × 106The mode of a/ml is suspended in comprising 10% passivation fetal calf serum (Cell Culture Technologies company), mould The culture of RPMI 1640 of plain 200U/ml, 200 μ g/ml of streptomysin, 0.01%L- glutamic acid (Life Technologies company) In base (Sigma-Aldrich company).The anti-ox LAG-3 antibody 2D8 of rat or the rat-ox that 10 μ g/ml are added into PBMC are embedding Anti- ox LAG-3 antibody ch2D8 is closed, at 37 DEG C, 5%CO2Under the conditions of cultivate 2 days.As control antibodies, rat blood serum is used Source IgG (Sigma-Aldrich company) and derived therefrom IgG (Sigma-Aldrich company).After 2 days, culture supernatant is recycled Liquid quantifies IFN-γ yield using Bovine IFN-γ ELISA Kit (BETYL company).In each of ELISA Automatic plate washer BIO WASHER 50 (DS Pharma Biomedical company) is used in cleaning operation, in absorbance Measurement in used microplate reader MTP-650FA (electric corporation CORONA).
Experimental result is shown in Fig. 6.Rat-ox inosculating antibody ox LAG-3 antibody ch2D8 and anti-ox the LAG-3 of rat is anti- Body 2D8 similarly makes the IFN-γ of ox PBMC respond rising.
2.11. the CDR analysis of the anti-ox LAG-3 antibody of rat
It uses NCBIIGBLAST (http://www.ncbi.nlm.nih.gov/igblast/), it is determined that the anti-ox of rat The complementary strand of LAG-3 antibody 2D8 determines area (CDR).It the results are shown in Fig. 1.
The application to other animal species of (embodiment 2) anti-lag-3 antibody
1. material, method and experimental result
1.1. the identification of sheep and buffalo LAG-3 gene
In order to determine buffalo (Bubalus bubalis；Asia buffalo) and the code area LAG-3cDNA (CDS) of sheep it is complete Long, design is first with ox and the base sequence (GenBank registration number AB608099 and XM_012129455) of sheep LAG-3 gene For the primer (buLAG-3CDS F and R, ovLAG-3CDS F and R) of the CDS overall length of basic amplification gene, with it is synthesized go out water Ox or sheep PBMC source cDNA have carried out PCR method as template.To resulting amplified production, surveyed according to well-established law using capillary Sequence instrument determines base sequence.
Primer (buLAG-3CDS F): ATGCTGTGGGAGGCTTGGTTC (sequence number 65)；
Primer (buLAG-3CDS R): TCAGGGATGCTCTGGCTGCA (sequence number 66)；
Primer (ovLAG-3CDS F): ATGCTGTGGGAGGCTCAGTTCCAGG (sequence number 67)；
Primer (ovLAG-3CDS R): TCAGGGTTGCTCCGGCTGCA (sequence number 68).
1.2. buffalo LAG-3 expresses the building of COS-7 cell
In order to make buffalo LAG-3 expression plasmid, using it is synthesized go out buffalo PBMC source cDNA as template, using to 5 ' The primer (buLAG-3-EGFP F and R) that end side is added restriction enzyme SacI and EcoRI recognition site and designs carries out PCR.After being handled using SacI and EcoRI (Takara company) resulting PCR product, extracted using FastGene Gel/PCR Kit (NIPPON Genetics company) purifying imports the pEGFP-N2 carrier for having carried out same restriction enzyme processing (Clontech company), is cloned.Use FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics company) expression plasmid is extracted, for being saved before testing with -30 DEG C.After, made expression plasmid is indicated For pEGFP-N2-buLAG-3.
Primer (buLAG-3-EGFP F):
ATTGAGCTCATGCTGTGGGAGGCTTGGTT (sequence number 69)；
Primer (buLAG-3-EGFP R):
AATGAATTCGGGATGCTCTGGCTGCAGC (sequence number 70).
By 5 × 104/cm2COS-7 cell passed in 6 orifice plates, including 10% passivation fetal calf serum 37 in 1640 culture medium of RPMI of (Invitrogen company), 0.01%L- glutamic acid (Life Technologies company) DEG C, 5%CO2In the presence of cultivate a Dinner.By pEGFP-N2-buLAG-3 or as the 0.4 μ g/cm of pEGFP-N2 of negative control2Make COS-7 cell is imported with Lipofectamine 2000 (Invitrogen company) and cultivates 48 hours (buLAG-3-EGFP tables Up to cell).In order to confirm the expression of the buffalo LAG-3 in produced expression cell, global function fluorescence microscope BZ- is utilized 9000 (KEYENCE companies) make the intracellular Cut-set power space of EGFP.
1.3. the reactivity (Fig. 7) of the anti-ox LAG-3 antibody 2D8 of rat and buffalo LAG-3
The anti-ox LAG-3 monoclonal antibody of rat is confirmed using Flow cytometry and cross reaction occurs for buffalo LAG-3. Buffalo LAG-3-EGFP expression COS-7 cell is had to the PBS of 10% passivation lowlenthal serum (Invitrogen company) using addition It closes 15 minutes at room temperature, reacts the anti-ox LAG-3 antibody 2D8 of the rat of 10 μ g/ml at room temperature 30 minutes, make after cleaning APC marks anti-rat Ig goat antibody (Beckman Coulter company) to react 30 minutes at room temperature.It is anti-as negative control Body has used rat IgG1 (κ) isotype controls (BD Biosciences company).FACS Verse has been used in analysis (BD Biosciences company).It should be noted that having used addition to have in the dilution of all cleaning operation and antibody The PBS of 1% bovine serum albumin(BSA) (Sigma-Aldrich company).
Experimental result is indicated with Fig. 7.It confirmed the anti-ox LAG-3 antibody 2D8 of rat and buffalo LAG-3 expression cell knot It closes.
1.4. the reactivity (Figure 10) of the anti-ox LAG-3 antibody 2D8 of rat and the lymphocyte of sheep
From the peripheral blood of sheep, the density-gradient centrifugation method for having used Percoll (GE Healthcare company) is utilized Isolate peripheral blood mononuclear cells (PBMC).The sheep PBMC isolated is suspended in comprising 10% passivation fetal calf serum (Invitrogen company), penicillin 200U/ml, 200 μ g/ml of streptomysin, 0.01%L- glutamic acid (Life Technologies company) 1640 culture medium of RPMI (Sigma-Aldrich company) in, be adjusted to 2 × 106A/ml.To Acetic acid Buddhist wave cardamom ester (phorbol 12-myristate acetate) (PMA) 20ng/ml is added in the PBMC and ion is mould Element (ionomycin) 1 μ g/ml (Sigma-Aldrich company), in 37 DEG C, 5%CO2Under the conditions of cultivate a Dinner.Recycling is cultivated PBMC, there is the PBS of 10% passivation lowlenthal serum (Invitrogen company) to close at room temperature 15 minutes using addition, make big The anti-ox LAG-3 antibody 2D8 of mouse reacts 30 minutes at 37 DEG C.As negative control antibody, rat blood serum source IgG has been used (Sigma-Aldrich company).After cleaning, using APC label goat anti-rat Ig antibody (Beckman Coulter company) into Line flag (room temperature, 30 minutes).Then, make the anti-sheep CD8 antibody of mouse (38.65, AbD Serotec company) anti-at room temperature It answers 30 minutes, is marked after cleaning using PerCP/Cy5.5 label goat anti-mouse IgG 2a antibody (Santa Cruz company) (room temperature, 30 minutes).Further after cleaning, Alexa Flour 488 is made to mark anti-sheep CD21 mouse antibodies (GB25A, VMRD Company) it reacts 30 minutes at room temperature.Label as GB25A has used Zenon labelling kit (Life Technologies company).FACS Verse (BD Biosciences company) has been used in analysis.It should be noted that In the dilution of all cleaning operation and antibody, addition is used to have 1% bovine serum albumin(BSA) (Sigma-Aldrich company) PBS.
By experimental result with Fig. 8 shows.The anti-ox LAG-3 antibody 2D8 of rat is activated with using the stimulation of PMA/ ionomycin Sheep CD8+T cell (CD21-CD8+Cell) and CD8-T cell (CD21-CD8-Cell；It include CD4+T cell and gamma delta T The cell masses of cell) it combines by force.
All publications, patent and the patent application quoted in this specification are used as without change and are incorporated by reference this theory In bright book.
Industrial availability
Anti-lag-3 antibody of the invention can be used for the prevention and/or treatment of the cancer of animal, infectious disease.
1 > of < sequence number
Sequence number 1 indicates the amino acid sequence of the L chain variable region of the anti-ox LAG-3 antibody of rat.Underscore portion: from the end NH2 It has held and has been followed successively by CDR1, CDR2, CDR3.
MMSPVQSLFLLLLWILGTNGDVVLTQTPPTLSATIGQSVSISCRSSQSLLDSDGNTYLNWLLQRPGQSP QLLIYSVSNLESGVPNRFSGSGSETDFTLKISGVEAEDLGVYYCMQATHVPFTFGSGTKLEIK
2 > of < sequence number
Sequence number 2 indicates the amino acid sequence of the H chain variable region of the anti-ox LAG-3 antibody of rat.Underscore portion: from the end NH2 It has held and has been followed successively by CDR1, CDR2, CDR3.
MVLLELVSVIALFQGVHCEVQLVESGGGLVQPKGSLRLSCAASGFDFDTYPMSWVRQAPGKGLDWVASI TIKTHNYATLYAASVKERFTISRDDSQSMVYLQMNNLKTEDTALYYCNREDFDYWGQGVMVTVSS
3 > of < sequence number
Sequence number 3 indicates the amino acid sequence of the L chain constant region (ox Ig lambda, GenBank:X62917) of Niu Kangti.
QPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWKADGSTITRNVETTRASKQSNSKYAASS YLSLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS
4 > of < sequence number
Sequence number 4 indicates the amino acid sequence of the H chain constant region (ox IgG1 changes GenBank:X62916) of Niu Kangti. Drawing in mutable site has underscore.Amino acid serial number and mutation: 119E → P, 120L → V, 121P → A, 122g → missing, 216A → S, 217P → S
ASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMV TVPGSTSGQTFTCNVAHPASSTKVDKAVDPTCKPSPCDCCPPPPVAGPSVFIFPPKPKDTLTISGTPEVTCVVVDVG HDDPEVKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPSSIVRTISRTKGPAR EPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADSSYFLYSKLRVDRNSWQE GDTYTCVVMHEALHNHYTQKSTSKSAGK
5 > of < sequence number
Sequence number 5 indicates the nucleotide sequence of the L chain variable region of the anti-ox LAG-3 antibody of rat.
ATGATGAGTCCTGTCCAATCCCTGTTTTTGTTATTGCTTTGGATTCTGGGAACCAATGGTGATGTTGTG CTGACCCAGACTCCACCCACTTTATCGGCTACCATTGGACAATCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCT CTTAGATAGTGATGGAAATACCTATTTAAATTGGTTGCTACAGAGGCCAGGCCAATCTCCACAGCTTCTAATTTATT CGGTATCCAACCTGGAATCTGGGGTCCCCAACAGGTTCAGTGGCAGTGGGTCAGAAACAGATTTCACACTCAAAATC AGTGGAGTGGAGGCTGAAGATTTGGGAGTTTATTACTGCATGCAAGCTACCCATGTTCCATTCACGTTCGGCTCAGG GACGAAGTTGGAAATAAAA
The codon-optimized rear nucleotide sequence of the nucleotide sequence of sequence number 5 is shown in 11 > of < sequence number.
ATGATGTCTCCCGTCCAAAGCTTGTTCCTGCTTCTCCTCTGGATTCTGGGCACAAACGGAGATGTGGTT CTCACCCAGACCCCCCCTACTCTGTCTGCCACCATCGGCCAGAGCGTGTCCATATCCTGTCGCAGCTCCCAAAGCCT GCTGGACTCCGATGGGAATACTTACCTGAATTGGCTGTTGCAGCGGCCTGGCCAGTCCCCCCAGCTGTTGATCTACA GCGTTAGCAATCTGGAAAGCGGGGTCCCCAACCGATTCTCCGGAAGCGGCTCCGAGACCGATTTTACCCTCAAGATC TCCGGCGTGGAAGCCGAGGACCTGGGAGTGTATTATTGCATGCAGGCCACCCATGTGCCCTTCACCTTCGGTAGCGG TACCAAGTTGGAGATCAAG
6 > of < sequence number
Sequence number 6 indicates the nucleotide sequence of the H chain variable region of the anti-ox LAG-3 antibody of rat.
ATGGTTCTCCTGGAGTTGGTTTCCGTGATTGCTCTTTTTCAAGGCGTGCATTGTGAGGTGCAGCTTGT TGAGTCTGGTGGAGGGCTGGTGCAGCCTAAGGGGTCATTGAGACTCTCATGTGCAGCCTCTGGATTTGACTTCGAT ACTTATCCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGTCTGGATTGGGTTGCTAGTATAACCATTAAGACTC ATAATTATGCAACACTTTATGCTGCTTCAGTGAAAGAGAGATTCACCATCTCCAGAGATGACTCACAAAGCATGGT TTACTTGCAAATGAACAACTTGA AAACTGAGGACACAGCCTTGTATTACTGTAACAGGGAGGACTTTGATTACTG GGGCCAAGGAGTCATGGTCACAGTCTCCTCA
The codon-optimized rear nucleotide sequence of the nucleotide sequence of sequence number 6 is shown in 12 > of < sequence number.
ATGGTGCTTCTCGAGCTGGTCAGCGTGATTGCTCTGTTTCAGGGCGTGCACTGCGAAGTGCAGCTGGTG GAGAGTGGTGGTGGGCTCGTGCAACCAAAAGGCAGTCTCAGGCTGAGTTGTGCCGCCTCCGGATTCGATTTCGACAC CTACCCAATGAGCTGGGTCAGGCAAGCCCCAGGGAAAGGACTCGATTGGGTGGCAAGCATTACCATCAAGACACACA ATTATGCTACCCTGTATGCCGCAAGCGTAAAGGAACGCTTTACCATCTCCCGCGATGATAGCCAGTCCATGGTATAT TTGCAAATGAATAATTTGAAGACAGAAGATACCGCTTTGTATTATTGCAACAGAGAAGATTTTGATTATTGGGGGCA GGGGGTGATGGTAACCGTGTCCAGC
7 > of < sequence number
Sequence number 7 indicates the nucleotide sequence of the L chain constant region (ox Ig lambda, GenBank:X62917) of Niu Kangti.
CAGCCCAAGTCCCCACCCTCGGTCACCCTGTTCCCGCCCTCCACGGAGGAGCTCAACGGCAACAAGGCC ACCCTGGTGTGTCTCATCAGCGACTTCTACCCGGGTAGCGTGACCGTGGTCTGGAAGGCAGACGGCAGCACCATCAC CCGCAACGTGGAGACCACCCGGGCCTCCAAACAGAGCAACAGCAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGA GCAGCGACTGGAAATCGAAAGGCAGTTACAGCTGCGAGGTCACGCACGAGGGGAGCACCGTGACGAAGACAGTGAAG CCCTCAGAGTGTTCTTAG
The codon-optimized rear nucleotide sequence of the nucleotide sequence of sequence number 7 is shown in 13 > of < sequence number.
CAGCCTAAGTCCCCTCCTTCAGTCACCCTGTTTCCACCATCTACCGAAGAACTCAACGGGAATAAAGC AACACTGGTGTGCCTTATTTCTGATTTTTACCCAGGGTCTGTGACAGTGGTTTGGAAAGCTGACGGTTCAACAATT ACAAGAAACGTGGAGACAACAAGGGCTTCTAAGCAGT CAAACTCTAAGTATGCTGCAAGTTCTTACCTTTCTCTT ACAAGTAGTGACTGGAAAAGTAAGGGCAGTTATTCATGCGAGGTCACTCACGAGGGAAGTACTGTAACTAAAACTGT AAAACCATCAGAGTGTTCATAG
8 > of < sequence number
Sequence number 8 indicates the nucleotide sequence of the H chain constant region (ox IgG1 changes GenBank:X62916) of Niu Kangti (after codon-optimized).
GCTAGCACCACAGCACCTAAAGTTTACCCTCTGTCTTCCTGCTGCGGCGACAAGTCTTCATCAACTGTT ACTCTTGGATGCCTGGTCTCAAGTTACATGCCCGAGCCCGTGACAGTGACCTGGAACTCAGGCGCTCTGAAGTCTGG AGTGCACACATTTCCAGCTGTGCTTCAGTCTAGCGGCCTGTATTCCCTCAGCTCTATGGTTACTGTACCTGGTAGCA CCAGCGGACAGACTTTCACCTGTAATGTTGCCCATCCCGCATCTTCTACCAAGGTCGATAAAGCCGTTGACCCCACT TGCAAACCATCCCCTTGTGATTGTTGTCCACCCCCTCCAGTGGCTGGCCCTTCCGTCTTCATTTTCCCTCCTAAACC TAAGGATACTCTGACCATCTCAGGGACACCCGAGGTCACCTGTGTCGTCGTGGACGTGGGACATGACGACCCAGAAG TCAAGTTCTCATGGTTCGTGGACGATGTGGAGGTGAACACAGCAACAACAAAGCCCAGAGAAGAACAGTTTAACAGC ACATATCGGGTGGTCAGCGCCTTGCGTATTCAGCACCAGGACTGGACTGGTGGCAAGGAGTTTAAGTGCAAGGTGCA TAACGAAGGTCTGCCCTCTTCTATAGTGAGAACTATCTCCCGAACTAAGGGCCCCGCTCGGGAGCCCCAGGTTTACG TCCTTGCTCCCCCTCAGGAGGAACTGAGTAAATCAACCGTGAGTCTCACCTGTATGGTTACCTCATTTTACCCAGAC TACATCGCCGTAGAGTGGCAGAGGAATGGACAGCCAGAGTCTGAGGACAAATACGGCACTACTCCTCCCCAACTGGA TGCCGACTCTTCCTACTTCCTCTACTCCAAATTGCGAGTTGACCGGAACTCATGGCAGGAGGGGGACACATACACAT GCGTCGTTATGCACGAGGCCCTGCACAACCATTACACCCAGAAGTCCACATCTAAAAGTGCAGGTAAGTAA
9 > of < sequence number
Sequence number 9 indicates the chimeric L comprising the L chain variable region of the anti-ox LAG-3 antibody of rat and the L chain constant region of Niu Kangti The amino acid sequence of chain.
MMSPVQSLFLLLLWILGTNGDVVLTQTPPTLSATIGQSVSISCRSSQSLLDSDGNTYLNWLLQRPGQS PQLLIY SVSNLESGVPNRFSGSGSETDFTLKISGVEAEDLGVYYC MQATHVPFTFGSGTKLEIKQPKSPPSVTL FPPSTEELNGNKATLVCLISDFYPGSVTVVWKADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCE VTHEGSTVTKTVKPSECS
10 > of < sequence number
Sequence number 10 indicates the H chain variable region comprising the anti-ox LAG-3 antibody of rat and the H chain constant region (ox of Niu Kangti IgG1, change GenBank:X62916) chimeric H chain amino acid sequence.
MVLLELVSVIALFQGVHCEVQLVESGGGLVQPKGSLRLSCAASGFDFDTYPMSWVRQAPGKGLDWVASI TIKTHNYATLYAASVKERFTISRDDSQSMVYLQMNNLKTEDTALYYCNREDFDYWGQGVMVTVSSASTTAPKVYPLS SCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPAS STKVDKAVDPTCKPSPCDCCPPPPVAGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTA TTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPSSIVRTISRTKGPAREPQVYVLAPPQEELSKSTVS LTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADSSYFLYSKLRVDRNSWQEGDTYTCVVMHEALHNHYTQK STSKSAGK
14 > of < sequence number
Indicate the core of the chimeric L chain of the L chain variable region comprising the anti-ox LAG-3 antibody of rat and the L chain constant region of Niu Kangti Nucleotide sequence (nucleotide sequence after codon-optimized).
ATGATGTCTCCCGTCCAAAGCTTGTTCCTGCTTCTCCTCTGGATTCTGGGCACAAACGGAGATGTGGT TCTCACCCAGACCCCCCCTACTCTGTCTGCCACCATCGGCCAGAGCGTGTCCATATCCTGTCGCAGCTCCCAAAGC CTGCTGGACTCCGATGGGAATACTTACCTGAATTGGCTGTTGCAGCGGCCTGGCCAGTCCCCCCAGCTGTTGATCT ACAGCGTTAGCAATCTGGAAAGCGGGGTCCCCAACCGATTCTCCGGAAGCGGCTCCGAGACCGATTTTACCCTCAA GATCTCCGGCGTGGAAGCCGAGG ACCTGGGAGTGTATTATTGCATGCAGGCCACCCATGTGCCCTTCACCTTCGG TAGCGGTACCAAGTTGGAGATCAAGCAGCCTAAGTCCCCTCCTTCAGTCACCCTGTTTCCACCATCTACCGAAGAAC TCAACGGGAATAAAGCAACACTGGTGTGCCTTATTTCTGATTTTTACCCAGGGTCTGTGACAGTGGTTTGGAAAGCT GACGGTTCAACAATTACAAGAAACGTGGAGACAACAAGGGCTTCTAAGCAGTCAAACTCTAAGTATGCTGCAAGTTC TTACCTTTCTCTTACAAGTAGTGACTGGAAAAGTAAGGGCAGTTATTCATGCGAGGTCACTCACGAGGGAAGTACTG TAACTAAAACTGTAAAACCATCAGAGTGTTCATAG
15 > of < sequence number
Indicate the H chain variable region comprising the anti-ox LAG-3 antibody of rat and H chain constant region (ox IgG1, the change of Niu Kangti GenBank:X62916 the nucleotide sequence (nucleotide sequence after codon-optimized) of chimeric H chain).
ATGGTGCTTCTCGAGCTGGTCAGCGTGATTGCTCTGTTTCAGGGCGTGCACTGCGAAGTGCAGCTGGT GGAGAGTGGTGGTGGGCTCGTGCAACCAAAAGGCAGTCTCAGGCTGAGTTGTGCCGCCTCCGGATTCGATTTCGAC ACCTACCCAATGAGCTGGGTCAGGCAAGCCCCAGGGAAAGGACTCGATTGGGTGGCAAGCATTACCATCAAGACAC ACAATTATGCTACCCTGTATGCCGCAAGCGTAAAGGAACGCTTTACCATCTCCCGCGATGATAGCCAGTCCATGGT ATATTTGCAAATGAATAATTTGAAGACAGAAGATACCGCTTTGTATTATTGCAACAGAGAAGATTTTGATTATTGG GGGCAGGGGGTGATGGTAACCGTGTCCAGCGCTAGCACCACAGCACCTAAAGTTTACCCTCTGTCTTCCTGCTGCG GCGACAAGTCTTCATCAACTGTTACTCTTGGATGCCTGGTCTCAAGTTACATGCCCGAGCCCGTGACAGTGACCTG GAACTCAGGCGCTCTGAAGTCTGGAGTGCACACATTTCCAGCTGTGCTTCAGTCTAGCGGCCTGTATTCCCTCAGC TCTATGGTTACTGTACCTGGTAGCACCAGCGGACAGACTTTCACCTGTAATGTTGCCCATCCCGCATCTTCTACCA AGGTCGATAAAGCCGTTGACCCCACTTGCAAACCATCCCCTTGTGATTGTTGTCCACCCCCTCCAGTGGCTGGCCC TTCCGTCTTCATTTTCCCTCCTAAAC CTAAGGATACTCTGACCATCTCAGGGACACCCGAGGTCACCTGTGTCGT CGTGGACGTGGGACATGACGACCCAGAAGTCAAGTTCTCATGGTTCGTGGACGATGTGGAGGTGAACACAGCAACAA CAAAGCCCAGAGAAGAACAGTTTAACAGCACATATCGGGTGGTCAGCGCCTTGCGTATTCAGCACCAGGACTGGACT GGTGGCAAGGAGTTTAAGTGCAAGGTGCATAACGAAGGTCTGCCCTCTTCTATAGTGAGAACTATCTCCCGAACTAA GGGCCCCGCTCGGGAGCCCCAGGTTTACGTCCTTGCTCCCCCTCAGGAGGAACTGAGTAAATCAACCGTGAGTCTCA CCTGTATGGTTACCTCATTTTACCCAGACTACATCGCCGTAGAGTGGCAGAGGAATGGACAGCCAGAGTCTGAGGAC AAATACGGCACTACTCCTCCCCAACTGGATGCCGACTCTTCCTACTTCCTCTACTCCAAATTGCGAGTTGACCGGAA CTCATGGCAGGAGGGGGACACATACACATGCGTCGTTATGCACGAGGCCCTGCACAACCATTACACCCAGAAGTCCA CATCTAAAAGTGCAGGTAAGTAA
16 > of < sequence number
Sequence number 16 indicates the amino acid sequence of the CDR1 of the L chain variable region of the anti-ox LAG-3 antibody 2D8 of rat (QSLLDSDGNTY)。
17 > of < sequence number
Sequence number 17 indicates the amino acid sequence of the CDR3 of the L chain variable region of the anti-ox LAG-3 antibody 2D8 of rat (MQATHVPFT)。
18 > of < sequence number
Sequence number 18 indicates the amino acid sequence of the CDR1 of the H chain variable region of the anti-ox LAG-3 antibody 2D8 of rat (GFDFDTYP)。
19 > of < sequence number
Sequence number 19 indicates the amino acid sequence of the CDR2 of the H chain variable region of the anti-ox LAG-3 antibody 2D8 of rat (ITIKTHNYAT)。
20 > of < sequence number
Sequence number 20 indicates the amino acid sequence of the CDR3 of the H chain variable region of the anti-ox LAG-3 antibody 2D8 of rat (NREDFDY)。
21 > of < sequence number
Sequence number 21 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG1 mutant 1).
22 > of < sequence number
Sequence number 22 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG1 mutant 2).
23 > of < sequence number
Sequence number 23 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG1 mutant 3).
24 > of < sequence number
Sequence number 24 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG2 mutant 1).
25 > of < sequence number
Sequence number 25 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG2 mutant 2).
26 > of < sequence number
Sequence number 26 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG2 mutant 3).
27 > of < sequence number
Sequence number 27 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG3 mutant 1).
28 > of < sequence number
Sequence number 28 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG3 mutant 2).
29 > of < sequence number
Sequence number 29 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG1 mutant 1).
30 > of < sequence number
Sequence number 30 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG1 mutant 2).
31 > of < sequence number
Sequence number 31 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG1 mutant 3).
32 > of < sequence number
Sequence number 32 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG2 mutant 1).
33 > of < sequence number
Sequence number 33 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG2 mutant 2).
34 > of < sequence number
Sequence number 34 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG2 mutant 3).
35 > of < sequence number
Sequence number 35 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG3 mutant 1).
36 > of < sequence number
Sequence number 36 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of Niu Kangti (IgG3 mutant 2).
37 > of < sequence number
Sequence number 37 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of sheep antibody (IgG1).
38 > of < sequence number
Sequence number 38 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of sheep antibody (IgG1).
39 > of < sequence number
Sequence number 39 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of sheep antibody (IgG2).
40 > of < sequence number
Sequence number 40 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of sheep antibody (IgG2).
41 > of < sequence number
Sequence number 41 indicates the amino acid sequence of L chain (Igkappa (CK)) constant region of sheep antibody.
42 > of < sequence number
Sequence number 42 indicates the nucleotide sequence of L chain (Igkappa (CK)) constant region of sheep antibody.
43 > of < sequence number
Sequence number 47 indicates the amino acid sequence of L chain (Ig lambda (CL)) constant region of sheep antibody.
44 > of < sequence number
Sequence number 44 indicates the nucleotide sequence of L chain (Ig lambda (CL)) constant region of sheep antibody.
45 > of < sequence number
Sequence number 45 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of buffalo antibody (being estimated as IgG1).
46 > of < sequence number
Sequence number 46 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of buffalo antibody (being estimated as IgG1).
47 > of < sequence number
Sequence number 47 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of buffalo antibody (being estimated as IgG2).
48 > of < sequence number
Sequence number 48 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of buffalo antibody (being estimated as IgG2).
49 > of < sequence number
Sequence number 49 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of buffalo antibody (being estimated as IgG3).
50 > of < sequence number
Sequence number 50 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of buffalo antibody (being estimated as IgG3).
51 > of < sequence number
Sequence number 51 indicates the amino acid sequence of L chain (the being estimated as Ig lambda) constant region (CL) of buffalo antibody.
52 > of < sequence number
Sequence number 52 indicates the nucleotide sequence of L chain (the being estimated as Ig lambda) constant region (CL) of buffalo antibody.
53 > of < sequence number
Sequence number 53 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of human antibody (IgG4 mutant 1).
54 > of < sequence number
Sequence number 54 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of human antibody (IgG4 mutant 1).
55 > of < sequence number
Sequence number 55 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of human antibody (IgG4 mutant 2).
56 > of < sequence number
Sequence number 56 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of human antibody (IgG4 mutant 2).
57 > of < sequence number
Sequence number 57 indicates the amino acid sequence of the H chain constant region (CH1~CH3) of human antibody (IgG4 mutant 3).
58 > of < sequence number
Sequence number 58 indicates the nucleotide sequence of the H chain constant region (CH1~CH3) of human antibody (IgG4 mutant 3).
59 > of < sequence number
Sequence number 59 indicates the amino acid sequence of the L chain constant region of human antibody.
60 > of < sequence number
Sequence number 60 indicates the nucleotide sequence of the L chain constant region of human antibody.
61~70 > of < sequence number
Sequence number 61~70 successively indicate primer boLAG-3-EGFP F, boLAG-3-EGFP R, boLAG-3-IgF, boLAG-3-IgR、buLAG-3CDS F、buLAG-3CDS R、ovLAG-3CDS F、ovLAG-3CDS R、buLAG-3-EGFP F, the nucleotide sequence of buLAG-3-EGFP R.
71 > of < sequence number
The amino acid sequence of the expression ox LAG-3 overall length of sequence number 71.
MLWEAWFQVWLFLQLLWAAAVEAPEPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLPRTRQVTWQHVP ESGSAAPTPRGPGPRRYTVLRLAPGGLRIGKLPLQPRVQLEEMGLQRGDFSLWLRPARRADAGEYHAAVRFGNRAL ACRLRLRVGQAAVTASPPGPLWTSSWVVLNCSFSRPDLPASVHWFRGPGRVPVQESPHHHLVGNFLFLPQVSSLDS GTWGCSLTYRDGFNVSITYNLAVLGLEPRATLTVYAGAGSKVELPCRLPPGVGIQSSLTAMWTPPGEGPDLLVAGD RNNFTLRLEAVGQAQAGTYTCRVHLQGRQLSATVTLAVITVTPKPYGSSGSLRKPFCEVTPASGQERFVWSPLDKR SQRRSPGPWLLTPDARPLSQPWQCHLYQGERLLGTAVYLTELSHPGAQRSGRALGAGRTAHLPLLILGLLFLLLLV TGASSFHLWRRQWRPRRFSALEHGTHPSQASSKTGELEPELEPEPDPEVEPEPEPEPESQPQLQPEQP*
72 > of < sequence number
Sequence number 72 indicates the amino acid sequence for being equivalent to the part of the 71st~No. 99 extracellular space of ox LAG-3.
GSAAPTPRGPGPRRYTVLRLAPGGLRIGK

Claims (18)

1. a kind of anti-lag-3 antibody, it includes (a) L chain and (b) H chain,
The L chain with the CDR1 comprising the amino acid sequence with QSLLDSDGNTY sequence number 16, with the amino acid of SVS It is dynamic other than the chain variable region L of the CDR3 of the CDR2 of sequence and the amino acid sequence with MQATHVPFT sequence number 17 and rat The L chain constant region of object antibody,
The H chain with the CDR1 comprising the amino acid sequence with GFDFDTYP sequence number 18, have ITIKTHNYAT sequence The H chain variable region of the CDR3 of the CDR2 and amino acid sequence with NREDFDY sequence number 20 of numbers 19 amino acid sequence and big The H chain constant region of animal's antibody other than mouse.
2. antibody according to claim 1, wherein L chain variable region and H chain variable region derive from rat.
3. antibody according to claim 2, wherein L chain variable region is the L chain variable region of the anti-ox LAG-3 antibody of rat, H Chain variable region is the H chain variable region of the anti-ox LAG-3 antibody of rat.
4. antibody according to claim 3, wherein L chain variable region has the amino acid sequence of sequence number 1, H chain variable region Amino acid sequence with sequence number 2.
5. antibody according to any one of claims 1 to 4, wherein the L chain constant region of the animal's antibody other than rat has There is the amino acid sequence of the constant region of Lambda chain or Kappa chain.
6. antibody according to any one of claims 1 to 5, wherein the H chain constant region of the animal's antibody other than rat has Have the amino acid sequence of the constant region of the immunoglobulin for the IgG4 for being equivalent to people or be imported into make ADCC activity and/or The mutation that CDC activity reduces.
7. antibody according to claim 6, wherein the animal other than rat is ox, and the L chain constant region of Niu Kangti has The amino acid sequence of the constant region of Lambda chain, and the H chain constant region of Niu Kangti has been imported into makes ADCC activity and/or CDC The mutation that activity reduces.
8. antibody according to claim 7, wherein the L chain constant region of Niu Kangti has the amino acid sequence of sequence number 3, The H chain constant region of ox antibody has the amino acid sequence of sequence number 4.
9. antibody described according to claim 1~any one of 8, four chain structures with two L chains and two H chains.
10. a kind of medical composition, it includes antibody according to any one of claims 1 to 9 as effective component.
11. medical composition according to claim 10 is used for the prevention and/or treatment of cancer and/or infectious disease.
12. medical composition according to claim 11, wherein cancer and/or infectious disease are selected from tumor disease, white blood Disease, chronic bacillary diarrhea, anaplasmosis, bacillary mazoitis, fungoid mazoitis, mycoplasma infection (such as mycoplasma mammary gland Inflammation, mycoplasmal pneumonia etc.), tuberculosis, small-sized piroplasmosis, Cryptosporidiosis, globidiosis, trypanosomiasis and leishmaniasis.
13. a kind of artificial gene DNA encodes the DNA of L chain and the DNA of (b ') coding H chain it includes (a '),
The L chain with the CDR1 comprising the amino acid sequence with QSLLDSDGNTY sequence number 16, with the amino acid of SVS It is dynamic other than the chain variable region L of the CDR3 of the CDR2 of sequence and the amino acid sequence with MQATHVPFT sequence number 17 and rat The L chain constant region of object antibody,
The H chain with the CDR1 comprising the amino acid sequence with GFDFDTYP sequence number 18, have ITIKTHNYAT sequence The H chain variable region of the CDR3 of the CDR2 and amino acid sequence with NREDFDY sequence number 20 of numbers 19 amino acid sequence and big The H chain constant region of animal's antibody other than mouse.
14. a kind of carrier, it includes the artificial gene DNA described in claim 13.
15. a kind of host cell is converted by the carrier described in claim 14.
16. a kind of manufacturing method of antibody, which includes host cell described in culture claim 15 and from culture The operation of anti-lag-3 antibody is acquired in object.
17. a kind of DNA encodes L chain,
The L chain with the CDR1 comprising the amino acid sequence with QSLLDSDGNTY sequence number 16, with the amino acid of SVS It is dynamic other than the chain variable region L of the CDR3 of the CDR2 of sequence and the amino acid sequence with MQATHVPFT sequence number 17 and rat The L chain constant region of object antibody.
18. a kind of DNA encodes H chain,
The H chain with the CDR1 comprising the amino acid sequence with GFDFDTYP sequence number 18, have ITIKTHNYAT sequence The H chain variable region of the CDR3 of the CDR2 and amino acid sequence with NREDFDY sequence number 20 of numbers 19 amino acid sequence and big The H chain constant region of animal's antibody other than mouse.
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